Note: Descriptions are shown in the official language in which they were submitted.
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1
ANTIMICROBIAL AGENT
The present invention relates to antimicrobial agents. More specifically, the
invention relates
to the antimicrobial activity of a series of anhydrofi-uctose derivatives.
Food degradation from various sources is recognised in the literature and
individual
chemicals are known which will inhibit one aspect or another of degradation
derived from
a single source. Degradation, and the loss of colour or flavour of freshly cut
plant parts
are known to be caused by oxidation, enzymes, microbes, and metal ions. For
example,
acidulants are known to prevent microbial degradation by maintaining a
relatively low pH
environment but their effectiveness is only temporary.
Salmonella, of which there are over two thousand different strains, is one of
the major
causes of food poisoning in humans. Salmonella is a genus of rod-shaped Gram-
negative
E~terobacte~iaceae that inhabit the intestine and cause infections such as
gastroenteritis
and typhoid. If invasive, they can cause enteric fevers (for example, typhoid
caused by
Salmonella typhi, or paratyphoid fever caused by Salmonella pa~atyphi). Other
strains of
Salmonella are associated with food poisoning (usually Salmonella Typhimurium,
Salmonella panama or Salmonella Enteritidis, the latter notorious for the
contamination
of poultry) and occasionally septicaemia in non-intestinal tissues.
It is well known in the art that Salmonella cannot propagate at pH values
below 4.5. As a
consequence, mildly acid products such as fine food and non-fermented meat
products are
especially susceptible to attack by Salmonella.
For meat products, nitrite is often used as a preservative. However, the
addition of nitrite
is restricted for toxicological reasons (due to its acute toxicity, together
with the dangers
associated with nitrosamine formation). As a result, Salmonella is only
inhibited at
concentrations of nitrite beyond 1,000 ppm, which are far beyond legal limits.
Instead, it has been shown that combinations of nitrite and sorbic acid can
increase the
effectiveness against Salmonella [Inhibition of Salmonella by Sodium Nitrite
and
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2
Potassium Sorbate in Frankfurters, Journal of Food Science, 47, 1982, p. 1615
ff].
Inhibition has been observed at concentrations beyond 50 ppm of nitrite
combined with
2600 ppm sorbic acid.
Other agents such as bacteriocins (Nisin) are unable to inhibit Salmonella in
food,
whereas benzoic acid is unsuitable because the inhibitory effect can only be
observed in
acid products. The inhibitory effect of phytogenic ingredients (or "natural
substances")
such as oil extracts from different spices, has also been tested, but again
the
concentrations required for achieving the inhibitory effect on Salmonella were
too high
and the sensorical influence on the food was too strong.
Thus, to date, the use of chemical substances has been severely limited
because on the
one hand they have to be safe from a toxicological view point, but on the
other hand they
must not influence the product sensorically.
The present invention seeks to alleviate the problems associated with prior
art chemical
substances and to provide new antimicrobial compositions based on
anhydrofructose
derivatives. In particular, the invention seeks to provide antimicrobial
agents that are suitable
for use in foodstuffs/feed.
In a first aspect, the invention provides an antimicrobial composition for use
against a micro-
organism selected from Listeria, Salmonella, Bacillus, Saccharomyces,
Pseudomonas,
Clostridium, Lactobacillus, Brochothrix, Micrococcus, Yersinia, Enterobacter
and
Zygosaccha~omyces, said composition comprising a cyclic compound having
Formula I,
R3
R~ R~ z/ Rs
R
I
or a derivative thereof,
wherein Rl and RZ are independently selected from -OH, =O, and OR', wherein R'
is H or -
COR", and R" is Cl_io alkyl;
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wherein R3 is a substituent comprising an -OH group;
wherein R4 and RS are each independently selected from a hydrocarbyl group, H,
OH or =O,
or represent a bond with an adj acent atom on the ring of the cyclic compound.
In a second aspect, the invention provides a process for preventing and/or
inhibiting the
growth of, and/or killing, micro-organisms in a material, the process
comprising the step of
contacting the material with a cyclic compound having Formula I,
R3
R~ R4 2/ Rs
R
I
or a derivative thereof,
wherein Rl and R2 are independently selected from -OH, =O, and OR', wherein R'
is H or -
COR", and R" is Cl_lo alkyl;
wherein R3 is a substituent comprising an -OH group;
wherein R4 and R5 are each independently selected from a hydrocarbyl group, H,
OH or =O,
or represent a bond with an adjacent atom on the ring of the cyclic compound;
wherein said micro-organism is selected from Listeria, Salmonella, Bacillus,
Saccharomyces, Pseudomonas, Clostridium, Lactobacillus, Brochothrix,
Micrococcus,
Yersinia, Euterobacter and Zygosaccharomyces.
In a third aspect, the invention relates to the use of a compound having
Formula I, or a
derivative thereof,
R3
R~ R4 z/ Rs
R
I
wherein R1 and RZ are independently selected from -OH, =O, and OR', wherein R'
is H or -
COR", and R" is Ci_lo alkyl;
wherein R3 is a substituent comprising an -OH group;
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wherein R4 and RS are each independently selected from a hydrocarbyl group, H,
OH or =O,
or represent a bond with an adj acent atom on the ring of the cyclic compound;
for preventing and/or inhibiting the growth of, andlor killing, micro-
organisms in a material,
wherein said micro-organism is selected from Listeria, Salmonella, Bacillus,
Saccharomyces, Pseudomonas, Clostridium, Lactobacillus, Brochothrix,
Mict~ococcus,
Yersinia, Enterobacte~ and Zygosaccharomyces.
Preferably, the material is a foodstuff or feed. Thus, in a preferred aspect,
the present
invention relates to antimicrobial substances that are suitable for use in
foodstuffs and/or
feed to inhibit food poisoning and spoiling bacteria contained therein.
By way of definition, the term "antimicrobial" refers to a substance that
kills or prevents or
inhibits the growth or reproduction of micro-organisms. Antimicrobials are
generally
classified according to the type of micro-organism they are effective against.
For example,
antibacterial substances are effective against bacteria, antifungal substances
are effective
against fungi, including yeast, and antiviral substances are effective against
viruses. Certain
antimicrobials can be used internally, for example antibiotic medications,
whereas other
antimicrobials are for external use only, such as antiseptics.
In a preferred aspect, the cyclic compound of the invention is a compound
having formula I,
or a derivative thereof,
wherein Rl and Ra are independently selected from -OH, =O;
wherein R3 is a substiiuent comprising an -OH group;
wherein R4 and RS are each independently selected from a hydrocarbyl group, H,
OH or =O,
or represent a bond with an adjacent atom on the ring of the cyclic compound.
In a more preferred aspect, the cyclic compound of the invention is a compound
having
Formula II
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R3
O
R5
R~ R4 R2
II
or a derivative thereof, wherein Rl, RZ, R3, R4, and RS are as defined above.
5 In a further preferred aspect, the cyclic compound of the invention is a
compound having
Formula III
R3
O
R5
R~ Ra R2
III
or a derivative thereof; wherein Rl, R2, R3, R4, and RS are as defined above.
In one preferred aspect of the invention the groups R4 and RS of the general
formula may
independently be a hydrocarbyl group.
The term "hydrocarbyl group" as used herein means a group comprising at least
C and
H and may optionally comprise one or more other suitable substituents.
Examples of
such substituents may include halo-, alkoxy-, vitro-, hydroxy, carboxyl,
epoxy, acrylic,
hydrocarbon, N-acyl, or cyclic group etc. In addition to the possibility of
the
substituents being a cyclic group, a combination of substituents may form a
cyclic
group. If the hydrocarbyl group comprises more than one C then those carbons
need
not necessarily be linked to each other. For example, at least two of the
carbons may
be linked via a suitable element or group. Thus, the hydrocarbyl group may
contain
hetero atoms. Suitable hetero atoms will be apparent to those skilled in the
art and
include, for instance, sulphur, nitrogen and oxygen.
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The groups R4 and RS of the general formula may independently be selected from
alkyl,
alkenyl, cycloalkyl and aryl or may together represent an alkylene.
In a particularly preferred aspect of the invention, the derivative of the
compound of
Formula I is an ester. The term "ester" includes mono-, di-, tri- and poly-
esters.
Preferably, the derivative of the compound of formula I is an ester wherein an
ester
linkage is formed from the -OH group of the R3 substituent. In this aspect
preferably the
derivatised R3 substituent is a group of the formula -(CHa)n OC(O)-(CHa)pCH3,
wherein n
and p are independently of each other from 1 to 24, preferably from 1 to 20,
preferably from
1 to 10, preferably from 1 to 5, or preferably l, 2, or 3. In yet a further
preferred
embodiment the derivatised R3 substituent is a group of the formula -CH2-OC(O)-
(CHa)pCH3, wherein p is from 1 to 24, preferably from 1 to 20, or p is from 1
to 10, or p is
from 1 to 5, and n=1, 2, or 3.
Preferably, the derivative of the compound of formula I is an ester wherein
the R1
substituent and/or the Ra substituent is an -OH group and wherein an ester
linkage is
formed from the -OH group of the Rl substituent and/or the R2 substituent. In
this aspect
preferably the derivatised Rl substituent and/or the R~ substituent is a group
of the formula
-(CH2)"-OC(O)-(CH2)pCH3, wherein n and p are independently of each other from
1 to 24,
preferably from 1 to 20, preferably from 1 to 10, preferably from 1 to 5, or
preferably l, 2, or
3. In yet a further preferred embodiment the derivatised Rl substituent and/or
the Ra
substituent is a group of the formula -CH2-OC(O)-(CH2)pCH3, wherein p is from
1 to 24,
preferably from 1 to 20, or p is from 1 to 10, or p is from 1 to 5, and n=1,
2, or 3.
Preferably, the derivative of the compound of formula I is an ester wherein
the R1
substituent and/or the R2 substituent is an -OH group and wherein an ester
linkage is
formed from the -OH group of the Rl substituent and/or the Rz substituent. In
this aspect
preferably the derivatised Rl substituent and/or the R2 substituent is a group
of the formula
-OC(O)-(CHZ)pCH3, wherein p is from 1 to 24, preferably from 1 to 20,
preferably from 1 to
10, preferably from 1 to 5, or preferably 1, 2, or 3.
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In a preferred aspect the compound of formula I is a diester wherein the Rl
substituent is
an -OH group and wherein the ester linkages are formed from the -OH group of
the R4
substituent and from the -OH group of the R3 substituent.
In a highly preferred aspect a derivative of the compound of formula I is a
compound of
the formula
\ //O O O
O
v ~O O
This compound (3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-enopyranose-
2-
ulose) may be prepared in accordance with the teaching of Andersen et al.
(1998),
Structure of 1,5-anhydro-D-fructose: X-ray analysis of crystalline acetylated
dimeric
forms, J. Carbohydr. Chem. 17: 1027-1035.
The aspect of the present invention wherein the derivative of the compound of
formula I
is an ester is particularly preferred because the compound may be lipophilic
and/or may
have both hydrophobic and hydrophilic properties. When the compound has both
hydrophobic and hydrophilic properties the compound readily resides at a
water/oil
interface of an emulsion.
The residence of the compound at a water/oil interface of an emulsion may
allow it to act
as an emulsifier. Thus the present invention may further provide compounds
having a
dual functional effect. The compounds may act both as an antimicrobial and as
an
emulsifier.
In particularly preferred aspect of the invention, the cyclic compound is
selected from
Ascopyrone P, Ascopyrone M, Ascopyrone T, Ascopyrone Tl, Ascopyrone T2, and
Ascopyrone T3, and mixtures thereof, the structures of which are shown below.
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HOCH2 HOCHz HOGHZ
0
HO 0 0 ~ OOH 0 O
Ascopyrone M Ascopyrone P Aseopyrone T
HOCH2 HOCH2 HOCHz
0 J---0 0
OH HO ~ HO HO
OH OH HO OH 0 ~ OH
Ascopyrone Ti Ascopyrone TZ Ascopyrone T3
Ascopyrone is a known compound. In 1978 and 1981, a group of American
scientists
prepared Ascopyrone P by pyrolysis of amylopectin, amylose and cellulose at
the Wood
Chemistry laboratory in Montana, with the intention of using Ascopyrone P as a
starting
material for organic synthesis [Shafizadeh, F., Furneaux R.H., Stevenson,
T.T., and
COChran, T.G., 1,5-Anhydro-4-de0xy-D-glycero-hex-1-en-3-ulose and other
pyrolysis
products of cellulose, CarbOhydr. Res. 67(1978): 433-447; Stevenson, T.T.,
Stenkmap,
R.E., Jensen, L.H., Cochran, T.T., Shafizadeh, F., and Furneaux R.H., The
crystal
structure of 1,5-anhydro-4-deoxy-D-glyeero-hex-1-en-3-ulose, Carbohydr. Res.
90(1981): 319-325]. They characterised Ascopyrone P by, for example, 1H and
13C NMR,
and IR spectroscopy techniques. A 3-dimensional structure of Ascopyrone P was
provided.
The yield of Ascopyrone P obtained by pyrolysis was under 3% and complicated
separation
methods had to be used.
The natural occurrence of Ascopyrone P in some species of very scarcely
studied fungi
collected from the Alps has been taught [M.-A. Baute, G. Deffieux, J.
Vercauteren, R.
Baute, and Badoc A., Enzymatic activity degrading 1,4-a-glucans to Ascopyrones
P and
T in Pezizales ad Tube~ales, Phytochemistry, 33 (1993): 41-45]. The occurrence
of
Ascopyrone P in fungi immediately prompted the hypothesis that Ascopyrone P
would act as
an antibiotic. However, Ascopyrone P did not function satisfactorily as an
antibiotic in the
disclosed tests.
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Many of the compounds of the present invention can be derived from 1,5-
anhydrofructose.
1,5-Anhydrofructose is monoketo sugar found in bacteria, red algae, fungi and
mammals.
In red algae and fungi 1,5-anhydrofructose is produced by the action of a-1,4-
glucan
lyase [EC 4.2.2.13] from floridean starch and glycogen, respectively.
When the compound of the present invention is prepared from 1,5-anhydro-D-
fructose,
preferably the 1,5-anhydro-D-fructose is prepared in accordance with GB-A-
2296717. In
other words, preferably the 1,5-anhydro-D-fructose is prepared by a method
comprising
treating an a-1,4-glucan with the enzyme a-1,4-glucan lyase characterised in
that enzyme is
used in substantially pure form.
Ascopyrone P and Ascopyrone T can be produced enzymatically from 1,5-anhydro-D-
fructose using cell-free extract prepared from the fungi of the order
Pezizales, such as
Plica~ia leioca~pa and Anthracobia melaloma, and the order of Tuberales, such
as, Tuber
melanosporum. Ascopyrone Tl is the dihydrate form of Ascopyrone T, whereas
Ascopyrone Ta and T3 are the tautomeric monohydrate forms of Ascopyrone T.
Ascopyrone M can be produced from 1,5-anhydro-D-fructose by EDTA-sensitive
dehydratases isolated from the fungi Morels, such as Morchella vulgaris,
Gyromitres,
pezizes, such as Peziza echinospora.
Ascopyrone M, P and T can also be produced chemically by treating 1,5-anhydro-
D-
fructose with alkali under mild conditions [Studies on the degradation of some
pentoses
and of 1,5-anhydro-D-fructose, the product of the starch-degrading enzyme a-
1,4-glucan
lyase; Thesis, Ahmad, T., The Swedish University of Agricultural Sciences,
Sweden,
1995].
When the compound of the present invention is prepared by chemical means, it
may be
prepared in accordance with one of the following methods:
(1) Ascopyrone P may be produced by treating 1,5-anhydro-D-fructose with non-
aqueous
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acid at elevated temperature, for example at 70 °C.
(2) Ascopyrones (for example, Ascopyrone P, T and M) may be produced from 1,5-
anhydro-D-fructose by alkaline treatment according to Ahmad, T., 1995.
5
The structures of all ascopyrones produced were confirmed by NMR techniques.
Preferably, the compound of the present invention is prepared by enzymatic
means as
disclosed in M.-A. Baute et al, [Phytochemistry, 33 (1993): 41-45). For
example
10 ascopyrones (such as, Ascopyrone P, T and M) may be produced from 1,5-
anhydro-D-
fructose using enzymatic methods as disclosed in M.-A. Baute et al.
In another preferred aspect, the cyclic compound of the invention is of
Formula IV,
R3
R~ Rs
R \R~
R ~R4
IV
or a derivative thereof,
wherein Rl and R2 are independently selected from -OH, =O, and OR', wherein R'
is H or -
COR", and R" is Cl_lo alkyl;
wherein R3 is a substituent comprising an -OH group;
wherein R4 and RS are each independently selected from a hydrocarbyl group, H,
OH or =O,
or represent a bond with an adjacent atom on the ring of the cyclic compound;
wherein R6 and R~ are each independently selected from H, OH or =O, or
represent a bond
with an adj acent atom on the ring of the cyclic compound.
In a more preferred aspect, the cyclic compound of the invention is of formula
V,
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R3 V
R7 0
s
R Rs
R Ra R2
or a derivative thereof, wherein R1, R2, R3, R4, R5, R6 and R' are as defined
above.
Even more preferably, the cyclic compound of the invention is selected from
one or more of
the following:
CHZOH CHZOH CHZOH
O O O
OH OH
OH OH ~ O
O OH O O
CHZOH CH~OH CHzOH
O O O
OH OH OH
OH OH OH~
HO OH OH O
O
CHZOH OAc O
P
O O O
OH
OH ~ OH
O OH Ac0 O p
p = 1,2....24
OH
O
O ~ \0H
In a particularly preferred aspect of the invention, R3 is or comprises a
CH20H group.
Preferably, the cyclic compound of the invention comprises a five or a six
membered ring.
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In a particularly preferred embodiment of the invention, the cyclic compound
having
Formula I has an antimicrobial effect against a micro-organism selected from
Listeria
mortocytogenes, Listeria ihnocua, Salmonella Typhimurium, Salmonella sp.,
Bacillus
cereus, Bacillus subtilis, Saccharomyces cerevisiae, Saccharomyces cerevisiae
var.
paradoxus, Saccharomyces carlsbergensis Pseudomorcas fl'uorescens, Clostridium
sporogehes, Lactobacillus sake, Brochothrix thermosphacta, Micrococcus luteus,
Yersinia
ehterocolitica, Ehterobacter aeroger~es, Zygosaccharomyces bailiff.
The cyclic compound of the invention may be used alone, or in combination with
other
components, for example, one or more chelators (such as EDTA sodium salt,
polyphosphate or citrate) and/or one or more antioxidants (such as ascorbate,
isoascorbate, ascorbate palinitate, BHA or BHT).
Tests indicate that APP is stable in water at 24 °C for 2 weeks, but
completely disappears
after 2 months. It has been reported that APP is stable at pH 1.5-5.5, and
less stable at
increasing pH. At pH 11-12.5 APP has a half life of 5 h. Since most food is
acidic or
neutral, the stability may therefore be improved by using APP in combination
with an
antioxidant such as those listed hereinbefore.
In one preferred embodiment, the compound is used in combination with one or
more
preservatives. By way of definition, in the broadest sense, the term
"preservative" is intended
to encompass all substances which inhibit the development of, or kill, micro-
organisms.
In a narrower sense, it is generally understood that preservatives are used in
concentrations of 0.5 °l° or less. Food additives which are
allowed to be used as
preservatives are listed in the Regulation No. 95/2/EG of the European
Parliament and
Council of 20 February 1995, relating to food additives other than colouring
agents and
sweeteners.
Typical food preservatives permitted in the EU which are suitable for use in
combination
with the compounds of the invention include sorbic acid, benzoic acid, PHB
ester (p-
hydroxybenzoate), and sulphur dioxide. The mode of action of these
preservatives,
together with their range of effects are listed below.
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Sorbic Acid (E200 to 203):
Mode of action: inhibits different enzymes in the cells of the micro-
organisms.
Range of effects: mainly against yeasts and moulds as well as catalase-
positive bacteria.
Catalase-negative bacteria as well as lactic acid bacteria and clostridia are
not inhibited.
Effective concentration: 500 - 3000 ppm.
Permitted maximum quantities in food: up to 2000 ppm in potato dough,
processed
cheese, packed bread, fine bakery products, emulsified sauces etc.
Benzoic Acid (E210 to 213):
Mode of action: inhibits exchange of oxygen through the cellular membrane and
affects
the enzymatic structure.
Range of effects: for acid products only, up to approx. pH 4.5; inhibits
yeasts and moulds,
restricted inhibition of bacteria (no, or only very little, inhibition of
lactic acid bacteria
and clostridia).
Permitted maximum quantities in food: 500 ppm in aspic, fruit preparations,
marmalades
etc.
PHB Ester (p-hydroxybenzoate) (E214 to 219)
Mode of action: damages the bacterial membrane because of the surface
activity,
poisonous to protoplasm because of protein denaturation.
Range of effects: mainly inhibits yeasts and fungi, but also Gram-positive
bacteria in a pH
range between 3.0 and 8Ø
Effective concentration: sensorical influence at concentrations beyond approx.
0.08 %.
Sulphur Dioxide (E220 to 224; E 226 to 227)
Mode of action: depends on pH to a great extent, in practice it is only
effective at acidic
pH values (< 4,0). Very complex mechanisms.
Range of effects: mainly antibacterial, above all against Gram-negative,
aerobic bacteria.
Effective concentrations: 250 - 500 ppm for inhibition of aerobic, Gram-
negative bacteria,
800 - 2000 ppm against Gram-positive bacteria, yeasts, and moulds.
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Permitted maximum quantity in food products: max. 2000 ppm in dry fruits,
grape juice
concentrate for home production of wine, in some cases only max. quantities of
20 - 30
ppm are permitted.
For more specific applications, the compounds of the present invention may
also be used
in combination with the following preservatives: biphenyl, diphenyl,
orthophenylphenol,
thiabendazol, raisin, natamycin, hexamethylentetramine, dimethyldicarbonate,
boric acid,
sodiumtetraborate, nitrite, propionic acid and propionate, and lysozyme. The
mode of
action of these preservatives, together with their range of effects and
specific uses are
listed below.
Biphenyl, Diphen~ (E 230)
Range of effects: Inhibition of moulds.
Substance for treatment of fruits: surface treatment of citrus fruits.
Permitted maximum quantity: 70 ppm
Orthophen~lphenol (E 231 / E 232)
As with E230, limited to treatment of fruits as a surface treatment for citrus
fruits.
Thiabendazol (E 233)
Surface treatment of citrus fruits and bananas.
Nisin (E 234)
Mode of action: Disturbance of membrane functions.
Range of effects: Gram-positive bacteria, no influence on Gram-negative
bacteria.
Permitted maximum quantity in food products (EU): 3ppm in semolina pudding and
similar products, 12.5 ppm (= 12.5 IU/g) in ripened cheese and processed
cheese, 10 ppm
in clotted cream, 10 ppm in mascarpone.
Natamxcin (Pimaricin) (E235)
Mode of action: specifically attacks cell membrane, where - in general - an
interaction
with sterines occurs which increases the permeability of the membrane.
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1S
Range of effects: Moulds and yeasts, not effective against bacteria. Usual
dosage rates are
below approx. 50 mg / 1. Maximum level is 1 mg/dma on the surface, with a
maximum
penetration of 5 mm.
Applications: surface treatment of hard, semi-hard and semi-soft cheese and of
dried,
cured sausages.
Hexamethylentetramine (E 239)
Hexamethylentetramine is formed by adding ammonia to formaldehyde in an
aqueous
solution. The microbicidal effect is due to the formaldehyde.
Permitted only for Provolone cheese (25 ppm residual quantity).
Dimethyldicarbonate (E 242)
Permitted only for non-alcoholic drinks, non-alcoholic wine, and liquid
concentrate.
Boric Acid, Sodiumtetraborate (E284 / E 285)
Permitted only for caviar.
Nitrite (E 249 and E 250)
Permitted in the form of nitrite curing salt for treatment of meat products
("red products").
For cured and dried meat products which are not heat treated and for other
cured meat
products an addition of 150 ppm has been fixed as a guideline. These
concentrations do
not show a preservative effect. They are mainly added for their technological
properties
(formation of colour, taste) as well as for their antioxidant effects.
Propionic Acid and Propionate (E 280, E 281, E 282, and E 283)
Mode of action: similar to sorbic acid, pH < 4.5 is optimal.
Accumulation in the cell leads to inhibition of enzymes.
Range of inhibition: moulds are inhibited at an pH of 5.5 by concentrations of
125 to
12500 ppm, for inhibition of bacteria higher concentrations are necessary (>
16000 ppm).
Application: Sliced and packaged bread.
Permitted maximum quantity: 3000 ppm.
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16
Lvsoz ie (E 1105)
Permitted only for ripened cheese.
Permitted maximum quantity: quantum satis.
Studies by the applicant of the inhibitive effects of the present compounds
have been
tested in a medium (Elliker broth) with an almost neutral pH (pH 6.8) and have
been
shown to be effective against both Gram-positive and Gram-negative bacteria.
As many
of the preservatives described above show an inhibitory effect mainly at low
pH, the use
of the compounds of the present invention clearly broadens the potential range
of
applications.
In principle, the use of substances for chemical preservation depends on the
following
factors:
(a) Toxicological harmlessness
~ the effects of the substance when applied acutely, subchronically, and for a
long term
period.
~ Testing of acute toxicity (LDso), cinetics and metabolism, pharmacological
effects,
genotoxicity, etc.
(b) Technological I food chemical aspects:
~ Solubility in water: as growth takes place in the aqueous phase, a
preservative has to
be water-soluble
~ Reaction with food ingredients, problem of off flavours (sensory acceptance)
~ Interferences with food ingredients (e.g. destruction of vitamin B1 by
sulphuric acid)
The antimicrobial effectiveness of chemical substances in food and feed
products is thus
determined by a range of different factors. Among others, the composition of
the
population of micro-organisms, the composition of the food product
(ingredients, pH,
water activity, content of salt, etc.), the packaging, time-temperature-
conditions, etc. are
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key factors that influence the inhibitory activities of the antimicrobial
agent.
The invention will now be described only by way of example, and with reference
to the
accompanying figures, wherein:
Figure 1 shows the antimicrobial activity of Ascopyrone P (APP) against
Salmonella Typhimurium DSMZ 554 (10e3 cfu/ml).
Figure 2 shows the antimicrobial activity of Ascopyrone P (APP) against
Salmonella Typhimurium DSMZ 554 (10e5 cfulml).
Figure 3 shows the effect of APP against S Typhimurium S29 at 30 °C
over a
period of 24 h.
Figure 4 shows the effect of APP on the growth of Br. thermosphacta CRA7883
over a 72h period.
Figure 5 shows the effect of APP on the growth of L. mor~ocytogenes 272 at 30
°C
over a period of 24 h.
Figure 6 shows the effect of APP on the growth of B. cereus 204 at 30
°C over a
period of 24 h.
Figure 7 shows the effect of APP on the growth of Ps. fluo~eseeus 3756 at 30
°C
over a period of 24 h.
Figure 8 shows the effect of APP on L. monocytogehes 272 in chicken soup at 8
°C
(detection limit 10a cfulg) over a 60 day period.
Figure 9 shows the effect of APP against Ps. fluorescens 3756 in chicken soup
at
8°C (detection limit 10a cfu/g) over a 49 day period.
Figure 10 shows the effect of APP against Salmonella Typhimurium S29
in chicken soup at 8 °C. Detection limit 102 cfu/ml. Trial finished at
49 d
Figure 11 shows the effect of APP against B. cereus 204 spores in chicken soup
at 8
°C. Detection limit 102 cfu/ml. Trial finished at 49 d
Figure 12 shows the effect of APP on S. Typhimurium S29 in a cooked mince beef
slurry system at 8°C.
Figure 13 shows the effect of APP against Salmonella Typhimurium S29 in minced
chicken slurry at 8 °C.
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EXAMPLES
1. PRELIMINARY INVESTIGATION
Inhibition of Gram-positive and Gram-native bacteria
Antimicrobial activity was investigated using Salmohella Typhimurium and
Listeria
annocua.
Bacterial growth was carried at 37°C for 24 hour for S Typhimurium and
20 hours for
Listeria ih~ocua, both at an inoculation size of l Oe3 or 10e5 cfu/ml (103 or
105 colony
forming units per ml).
In both cases, APP showed a satisfactory inhibitory effect.
S. Typhimurium
For S. Typhimurium at 10e3 and 10e5, complete inhibition was observed at 2000
~.g/ml and 4000 ~g/ml (see Figures l and 2). Intermediate inhibition was
observed at
500 wg/ml, with strong inhibition observed at 1000 ~glml.
L. iu~cocua
For L. innocua at 10e3, almost complete inhibition was observed at 2000 ~g/ml,
intermediate inhibition at 1000 ~,g/ml, and slight inhibition at 500 ~,g/mL At
10e5,
intermediate inhibition was seen at 2000 ~,g/ml, slight inhibition at 1000
~g/ml, and
almost no inhibition at 500 ~g/mL
The tests described above were all performed under the optimal growth
conditions for the
bacteria at high inoculation levels. In food, the growth conditions for these
bacteria will
be less favourable and the inoculation level will be lower, therefore lower
dosages will be
needed to inhibit their growth. In other words, lower concentrations of the
compounds of
the invention will be necessary in food products, for a sufficient inhibition
of food
poisoning or food spoiling micro-organisms.
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The above results clearly show that APP inhibits both Gram-negative bacteria
of the
Ehterobacte~iaceae type (Salmonella Typhimurium) and Gram-positive bacteria
(Listeria ihnocua).
Further trials were undertaken to investigate the antimicrobial efficacy of
ascopyrone P
(APP) in laboratory media and in a range of food systems.
2. MATERIALS AND METHODS
2.1 TEST STRAINS
All micro-organisms were taken from storage at -80 °C. Most organisms
were tested as
vegetative cell suspensions from overnight broth culture. Bacillus and
Clostridium species
were tested as endospore suspensions prepared earlier and stored at 4
°C.
For broth cultures and Bioscreen testing most bacteria were grown in Brain
Heart
Infusion (BHI, Oxoid, pH 7.4). Lactobacillus sake A10 was grown in de Man,
Rogosa,
Sharpe medium (MRS, Oxoid). Yeasts were grown in Sabouraud Liquid medium (SLM,
Oxoid). Most bacteria were cultured at 30 °C. Lactic acid bacteria were
grown on solid
medium in enriched COZ atmosphere. Clostridium species were grown in
Reinforced
Clostridial Medium (RCM) at 37 °C anaerobically. Brochothrix
thermosphacta, fungi and
yeasts were grown at 25 °C. Fungi were cultured on Malt Extract Agar
(MEA, Oxoid).
2.2 ASCOPYRONE P (APP) SAMPLES
Sample E002012 was a dry powder that was dissolved in sterile de-ionised
water. It was
tested without filtration and used in the mini well diffusion test, initial
Bioscreen runs
(BS1211200; BS131200), and mini cidal experiment. The concentration of this
sample
was 49.3 mg/ml. Further APP samples were prepared as follows: 3.84 g of batch
number
APP20010213, as 5 x 4 ml volumes, was made up to a concentration of 169 mg
APP/ml,
and 5.5 g of batch number APP20010215, as 4 x 10 ml volumes, was made up to a
concentration of 138 mg APP/ml. All samples were kept at -20 °C until
use. Stock
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solutions were made up in de-ionised water and filter sterilised. These
samples were used
in Bioscreen run BS010411 and BS010420. APP20010213 was used in the following
trials: chicken soup at 8 °C (TCS010412; TCS 010430); raw meat
(TRM010521).
APP20010215 was used in Bioscreen run BS010510, Clostridium sensitivity tests
and in
5 the following trials: chicken soup at 8 °C (TCS010521); apple juice
at 25 °C
(TAJ010530); milk (TMBG010605), sausage meat slurry test (TSM010605); further
raw
meat tests (TRM010620) and cooked beef and chicken slurry tests (TCM010619;
TCC010704).
10 2.3 IN VITRO GROWTH INHIBITION TEST METHODS TESTING
2.3.1 Well diffusion testing
Seeded 10 ml agar plates of various test organisms were prepared, with
inoculation of
either 20 p,1 spore suspension or overnight broth. This gave an inoculum level
of ca. 105 -
15 106 cfu/ml. After the plates had set, small wells were cut, and 20 ~.l of
APP sample was
loaded. Plates were incubated overnight at appropriate temperature and
examined after 1-
2 days (after which microbial growth is clearly visible) for zones of
inhibition.
2.3.2 Bioscreen testing
20 An automated Microbiology Reader Bioscreen C was used to measure growth
curves of
the strains in the presence and absence of APP. The Bioscreen C measures the
development of turbidity (i.e. growth) kinetically by vertical photometry in
200 wells of a
honeycomb microtitre plate, simultaneously. The system consists of a Bioscreen
C
analyser, which is an incubator and measurement unit, integrated with a PC,
software
(BioLink v 5.30), printer and a 'Honeycomb 2' cuvette multiwell plate. Growth
curve
data can be analysed within the BioLink software or exported to programs such
as Excel.
Broth culture media were dispensed in 270 ~,l volumes into the wells as
directed. Serial
dilutions of a filter-sterilised APP stock solution were then dispensed into
the same wells,
as appropriate. The wells were inoculated with 30 ~1 of an appropriately
diluted overnight
broth culture or spore suspension, to give a final inoculum level of ca. 103
cfu/ml. The
tests were incubated in the Bioscreen C for either 24 h at 30 °C, or 72
h at 25 °C
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(depending on the micro-organism under investigation) with readings taken
every 20
minutes after the trays were shaken. After the incubation period was complete
the data
were exported to Excel for analysis.
2.3.3. Sensitivi testin for Clostridium species
Stock solutions of APP215 (0.5, l and 2%) were prepared and filter-sterilised.
Cooked
meat medium (CMM, Oxoid) was prepared by distributing 1 g of the medium to
individual test tubes, which were then filled with 8.9 ml water. After
autoclaving 100 ~,1
of the APP stock solutions were added, to give final concentrations of 0, 500,
1000 and
2000 ppm APP. The tests were inoculated with 100 w1 of Clostridium spores, to
give an
inoculum level of ca. 103 cfulml. The tubes were overlaid with 3 ml of 2% agar
to
maintain anaerobicity. The tests were incubated at 37 °C, and examined
daily for signs of
growth (turbidity, gas production).
2.4. IN VITRO CIDAL EXPERIMENT
100 ~.l of SDW (sterile deionised water) or APP sample E002012 was added to
890 p.1 10
mM HEPES buffer (pH 7). 10 ~.1 of an overnight culture of L. monocytogenes S23
was
added to the test, which was incubated at ambient temperature for 2 h. After
this time the
bacteria were enumerated by viable count. The concentration of the APP sample
was 24.7
~.g/ml.
2.5 IN VIVO (FOOD) GROWTH INHIBITION TESTS
2.5.1 Food testing in chicken soup
A local supermarket brand of cream of chicken soup was used as a model food
system.
600 g packs were purchased from the local supermarket. This was a 'fresh home-
made
type' of soup that was stored in the chill cabinet, with a short shelf life.
It was chosen
because it is a rich, nutritious food system containing a range of food
components
including meat, dairy and vegetable.
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Ingredients were labelled as: chicken stock (water, duck fat, chicken, salt,
flavourings,
yeast extracts, pork gelatine, glucose syrup, vegetable concentrate, milk
protein, sugar,
malto-dextrin, vegetable oil, citric acid, lactose, chicken fat), milk,
chicken (9%), cream
(8%), onion, potato, vegetable oil, modified maize starch, wheat flour, lemon
juice, salt,
white pepper. pH 5.912. Protein: 3.3%; carbohydrate: 4.7%; fat: 7.2%; fibre:
0.2%;
sodium: 0.1
For the test the soup was mixed 2:1 in water (to facilitate sampling and
mixing) and
sterilised by autoclaving at 121 °C for 15 minutes. For each experiment
the pH of the
soup was recorded; this was between pH 5.79-5.88.
For TCS010412/8 (which was incubated at 8 °C), APP batch number
APP20010213
(concentration 169 mg APP/ml) was used to prepare tests containing 0, 2000 or
4000 ppm
APP. 5 g of filter-sterilised APP stock solution was added to 94 g of soup,
mixed and then
1 ml of a diluted inoculum suspension (cells or spores as appropriate) was
added to give a
test inoculum level of 103 cfu/g. The soup mixture was shaken vigorously to
ensure
homogeneous distribution of the APP and micro-organisms. The tests were tested
by
viable count and then incubated at 8 °C and tested on a twice-weekly
basis.
For TCS010430/20 (incubated at 20 °C) samples were prepared in 18.8 g
quantities, to
which 1 g of a suitable filter-sterilised APP stock solution was added and an
appropriately
diluted 0.2 ml inoculum. Great care was again taken to ensure homogeneous
distribution
of the APP and micro-organisms. This test was sampled by viable count test on
a daily
basis.
For TCS010521/8 (incubated at 8 °C), samples were prepared in 27.8 g
quantities. 1.5 g
of an APP stock solution was added, and the tests inoculated with 0.3 ml of a
suitably
diluted cell or spore suspension to give a final inoculum of ca. 103 cfu/g.
APP was tested
at 0, 500 and 1000 ppm, and batch number APP20010213 (at 169 mg/ml) was used.
The
tests were sampled by viable count and then incubated at 8 °C and
tested on a twice-
weekly basis.
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2.5.2 Test in apple 'uice~TAJ010530/25],
8.9 ml apple juice samples (pH 3.42) were prepared and autoclaved. Batch
number
APP20010215 (at 138 mg/ml) was used to prepare filter-sterilised stock
solutions for 1 ml
addition to the juice to achieve 0, 1000, 2000 ppm APP. Tests were inoculated
with 100
~,1 of an appropriately diluted overnight SLM broth of yeast culture or
ascospore
suspension of Byssochlamys spores prepared earlier. The tests were incubated
at 25 °C,
and examined daily for signs of growth (e.g. turbidity, mycelium development).
2.5.3 Test in milk jTMGB0106051
8.9 ml samples of UHT milk containing 0.5% glucose and the indicator
bromocresol
purple (0.01%) were prepared and sterilised by autoclaving. The pH before APP
addition
was pH 6.65. Appropriate stock solutions of APP batch number APP20010215 (at
13 8
mg/ml) were prepared and filter sterilised. 1 ml of each was added to the milk
to prepare
tests containing 0, 1000 and 2000 ppm APP. Tests were then inoculated with 100
p,1 of
diluted overnight broth cultures or spore suspensions to give a final inoculum
level of ca.
103 cfu/ml. These and uninoculated tests were incubated at 20 and 8 °C
and examined
daily for visible changes compared to uninoculated control.
2.5.4. Test in cooked sausage meat slurry [TSM0106051
500 g of bologna sausage meat mixture was mixed with 500 g Brain Heaxt
Infusion broth
(BHIB) to make a homogeneous slurry. The bologna sausage meat mix comprised (g
per
kg): lean beef 320; lean pork 202; lard 250; water 40; glucose 5; starch 155;
spice mix
3.75; sodium glutamate 0.5; sodium erythorbate 0.545; sodium di-phosphate 3;
NaCI 20;
sodium nitrite 0.15. 200 g samples of the meat slurry were autoclaved. Whilst
the meat
was still warm it was re-mixed thoroughly. When the meat had cooled it was
inoculated
with 2 ml of 105 cfu/ml spore or cell suspension, and re-mixed again. Each
inoculated
meat sample was divided into 3 x 50 g samples, and 1 ml of either SDW, 2.5% or
5%
APP solution (batch number APP 20010215) was added to prepare meat samples
containing 0, 500 and 1000 ppm APP. The meat slurry was again shaken
thoroughly to
ensure even mixing, an initial viable count was taken and the samples
incubated at 8 °C.
Samples were taken at twice-weekly intervals. The pH of the sausage meat
slurry was 6.2.
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2.5.5. Test in cooked minced beef slurr,~[TCM010619]
1 kg of minced beef, purchased from the local butcher, was mixed with 1 litre
of BHI.
200 g portion of this meat slurry was autoclaved. When the meat had cooled,
the pH was
recorded (pH 6.24), and each pot was inoculated with 2.0 ml of 105 spores or
cells and the
meat re-mixed thoroughly. Each inoculated 200 g pot was distributed as 3 x 50
g portions.
To each pot 1 ml of SDW, 5% or 10% APP (Batch No. APP20010215) was added, and
the meat was again mixed thoroughly. An initial viable count was performed,
the meat
was incubated at 8 °C and sampled twice weekly. The pH of the cooked
minced beef
slurry was 6.24.
2.5.6. Cooked minced chicken slurry [TCC0107041
1 kg of minced chicken (mainly lean meat) was purchased from the local
butcher. This
was mixed with 1 litxe of BHI, mixed and dispensed as 100 g samples prior to
autoclaving. When still warm, the meat was re-mixed thoroughly. When cooked,
the meat
was inoculated with 1 ml of 105 spores or cells (from an overnight broth), and
re-mixed.
The samples were then divided into 3 x 25 g portions, to which 0.5 ml of
filter-sterilised
APP20010215 was added (as 5% or 10% stock solutions) or water. An initial
viable count
was taken, and the samples then incubated at 8 °C and tested twice
weekly. The pH of the
chicken slurry was 6.42.
2.5.7. Uninoculated test in raw meat~TRM0105211
300 g of raw minced beef was purchased from the local butcher. 10 g of either
a 2%
(20,000 ppm) stock solution of APP2001213 or water was added, and the meat
mixed
thoroughly. Initial counts were carried out, and then the meat was incubated
at 8 °C and
sampled for 3 consecutive days. The pH of the raw meat was 5.3.
2.5.8. Inoculated test in raw meat test [TRM0106201
Fresh beef steak was purchased from the local butcher, and minced on clean
machinery.
The raw meat (ca. pH 6.2) was divided into 8 x 90 samples and inoculated-with
103
cfu/ml of test organisms. The meat was mixed thoroughly using a stomacher. 10
g of
SDW or 2% APP (Batch number APP20010215) was added, and the meat was again
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thoroughly mixed. An initial viable count was taken, the meat was incubated at
8 °C, and
sampled on the following days using non-selective agar (MPCA) and selective
agar: XLD
for Salmonella; Oxford selective agar (Listeria monocytogenes); and STAA agar
(Brochothrix thermosphacta). The pH of the raw meat was pH 5.3.
5
3.RESULTS
3.1. IN VITRO GROWTH INHIBITION RESULTS
3.1.1. Well diffusion results
10 The concentration of the test solution (unknown at time of testing) was
49.3 mg/ml
(4.9%). All strains tested were sensitive to this level. They included:
Bacillus cereus 204;
Clostridium sporogenes Carapden; Listeria mo~ocytogenes 523; Micrococcus
luteus;
Lactobacillus sake A10; Brochothrix thermosphacta CRA7883; Pseudomonas
fluorescens 327; Saccharomyces carlsbergensis CRA6413; Saccharomyces
cerevisiae
15 ATCC 9763.
3.1.2. Bioscreen test results: iu vitro sensitivity of bacteria and yeasts
The Bioscreen test results are summarised in Tables 1-4 and Figures 3-7.
Bioscreen run
BSO10510 was interrupted by power failure due to electrical storm overnight.
3.1.3. Sensitivity of Clostridium species to APP
No growth inhibition was observed at the highest level tested (2000 ppm
APP20010215)
against four strains of Cl. sporoger~es (strains 1.221; Carapden; ABC20;
4.440) and one
strain of Cl. tyrobutyricum 2753 grown in cooked meat medium anaerobically at
37 °C.
3.2. IN VITRO GIDAL TEST RESULTS
After 2 h incubation at ambient temperature in buffer containing 24.7 ~,g/ml
APP, the
Listeria counts remained steady, (Table 5).
3.3. IN hIVO (FOOD) GROWTH INHIBITION TEST RESULTS
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3.3.1. Results of chicken soup trials
The results of the chicken soup experiments incubated at 8 °C
[TCS010412, TCS010521]
and 20 °C [TCS010430] are shown in Tables 6-8, and Figures 8-11. The
APP had no
effect on the colour of the product.
3.3.2 Results of trials in apple juice incubated at ambient temperature
[TAJ0105301
1000 and 2000 ppm APP controlled the growth of Zygosaccha~omyces bailiff
CRA229 for
3-4 days. 2000 ppm controlled the growth of S cerevisiae ATCC9763 for 1 day.
No
growth inhibition was achieved by 1000 or 2000 ppm APP against Candida
pa~apsilosis
458; Hanseniaspora uvarum CBS5074; Rhodotorula mucilagihosa var mucilagiuosa
CBS8161; and the heat-resistant fungus Byssochlamys fulva 040021, and B. uivea
163642.
3.3.3 Results of milk trials incubated at 8 and 20°C [TMGB0106051
At 20 °C results were as follows:
No control observed at 2000 ppm for L. monocytogenes, Br. thermosphacta, Lb.
sake.
Control for 2- 3 d at 2000 ppm for Ps. fluorescens
Control for 4 d at 1000 ppm, and 6 d at 2000 ppm for B. cereus
At 8 °C results were as follows:
No control observed at 2000 ppm for L. mouocytogenes, Lb. sake, B. cereus
Control for 1 d at 2000 ppm for Br. thermosphacta
Control for > 7 d at 2000 ppm for Ps. fl'uoresceus
3.3.4. Results of trial in cooked sausage meat slurry at 8 °C [TSMOl
0605]~pH 6 2)
Results are summarised in Table 9.
3.3.5. Results of trial in cooked minced beef slurry~TCM0106191
Graphs of growth curves are shown in Figure 12 and summarised in Table 10.
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3.3.6. Results of trial in cooked minced chicken slurry at 8 °C
~TCC0107041
Growth curves are shown in Figure 13, and summarised in Table 11.
3.3.7. Results of uninoculated trial in raw meat at 8 °C [TRM0105211
(pH 5.3~
Results are shown in Table 12: counts were identical in the control and APP
samples. No
negative effect of APP on the meat colour was observed.
3.3.8 Results of inoculated trial in raw meat at 8 °C [TRM0106201
Despite every precaution the TAVC of the meat was high; 106 cfu/g rising to >
10' cfulg
by the end of the trial. No significant effect of APP was observed. No
negative effect was
observed on the meat colour.
4.0 CONCLUSIONS
The results of the above tests are summarised in Table 13. The tests indicate
that 2000
ppm APP is efficacious against the following micro-organisms: B. cereus, L.
monocytogehes, Ps. fluorescens, Salmonella Typhimurium, Lb sake, Y.
ehtef~ocolitica, Br.
thermosphacta, Z bailiff and S. cerevisiae.
The observed inhibitory activity is probably bacteriostatic, but in the
presence of
additional preservative hurdles (particularly low temperature), a cidal effect
is possibly
achieved with time. Against some bacteria, efficacy of APP was better in a
food system
compared to in laboratory media, probably because the organism grew less well
in the
food. The efficacy of APP may vary with different foods due to the differences
in their
chemical compositions. For example, APP was less efficient in a sausage meat
model
than in chicken soup.
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5.0 Tabulated Data
Table 1: Bioscreen results: effective levels of APP against Gram-positive
bacteria
GRAM POSITIVE APP
LEVEL
(ppm)
CAUSING
SIGNIFICANT
OR
TOTAL
INHIBITION
FOR
T1ME/TEMPERATURE
INDICATED
[Bacillus testedBSO10510 BS010411 BS010411
as 24 24 24
spores] h h h
at at at
30 30 30
C. C. C.
[APP20010213] [APP20010213] APP20010215]
TotalSi ificantTotal Si ificantTotal Si ificant
B. cereus 204 > 40004000 4000 2000
B. cereus Cam 4000 2000 > 40002000
den
B. cereus 3.046 30002000
B. subtilis Cam 30002000
den
L. monoc to eves3000
358
L. monoc to enes 4000 2000 >4000 2000
272
L. monoc to eves 4000 2000 4000 1000
S23
L. monoc t. Scott30002000
A
L. monocyt. F686130002000
Lb. sake A10 500 250 500 250
Table 2: Bioscreen results: effective levels of APP against Br.
tlzermosplzacta
BS010420
72 h
at 25
C
[APP20010213
APP
( m)
Total Si ificant
Br. thermosphacta4000 2000
CRA7883
Table 3: Bioscreen results: effective levels of APP against Gram-negative
bacteria
APP
LEVEL
(ppm)
CAUSING
SIGNIFICANT
OR
TOTAL
INHIBITION
FOR
TIME/TEMPERATURE
INDICATED
GRAM APP20010213 APP APP
20010213 20010215
NEGATIVES BSO10510 BS010411 BS010411
24 24 24
h/30 h/30 h/30
C C
Total Si ificantTotal Si ificantTotal Si ificant
Ps. fluorescens 2000 1000 2000 1000
3756
Ps. fluorescens2000 1000
10460
Ps. fluorescens1000
1331
Ps. uorescens1000
327
Ent. aerogenes4000
10102
Y. enterocolitica2000
S16
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Salm. 4000 2000 > 4000 2000
T himurium
S29
Salmonella 4000
s . S19
Table 4: Bioscreen results: effective levels of APP against yeasts
YEASTS APP 213
BS010420
72 h/25
C
Total Si ificant
S. cerevisiae ATCC9763> 4000 Enhanced owth
S. carlsber ensis > 4000 Enhanced owth
CRA6413
S cerevisiae H78 > 4000 4000
S cerevisiae var. > 4000 2000
paradoxus
H103
Table 5: Results of tidal experiment with APP E002012
Strain 0 pg/ml APP 24.7 ~,g/ml APP
L, mor~ocytogenes S23 4.3 x 10 1.3 x 10
Table 6: Chicken soup incubated at 8 °C. [TCS010412] APP20010213
tested at 0,
2000, 4000 ppm
Strain Result
B, tarsus 204 _
Total inhibition by 2000 and 4000 ppm
for > 60 days.
Counts remain at initial level , i.e.
no s ore kill.
L. monocytogenes Total inhibition by 4000 ppm for > 60
272 days.
Counts reach < 105 with 2000 ppm.
Counts undetectable after 20 days at
4000 ppm
Counts undetectable after ca 50 da s
at 2000 m
Ps. fluorescens Total inhibition by 2000 and 4000 ppm
3756 for > 60 days.
Counts undetectable from da 1 in APP
sam les
Table 7: Chicken soup at 8 °C [TCSM010521]. APP215 tested at 0,
500,1000 ppm
Strain Result
B. tarsus 204 Total inhibition by 500 and 1000 ppm for
> 27 days.
Counts remain at a rox initial level ,
i.e. no s ore kill.
L. monocytogenes No total inhibition by 1000 ppm, but slight
272 reduction of
growth rate
No effect with 500 m
L. monocytogenes No total inhibition by 1000 ppm
S23
Sli ht reduction of owth rate with 500
and 1000 m
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Ps. fluorescens . Total inhibition by 1000 ppm for > 27
3756 days.
At 1000 ppm counts undetectable > 3 d
Little effect with 500 m
Ps, fluorescens Total inhibition by 1000 ppm for > 27
327 days.
At 1000 ppm counts undetectable > 7 d
Little effect with 500 m
Salmonella S 19 Total inhibition by 500 -1000 ppm for
22 d
Slow owth in controls, reached 106 b
12 d
Salm Typhimurium Total inhibition by 1000 ppm for > 27
S29 d
Total inhibition by 500 ppm for 25 d
Slow owth in controls, reached 106 b
14 d
S. cerevisiae 9763 No effect with 500 and 1000 ppm
Table 8: Chicken soup incubated at 20 °C [TCSM010430]. APP20010213
tested at 0,
200, 500,1000, 2000 ppm
Strain Result
B. cereus 204 Total inhibition by 2000 ppm for > 42 days.
Extension of lag with 1000 ppm
No si ificant effect with 500 m
L. monocytogenes No total inhibition but reduction of growth
272 rate only by 2000
ppm
No si ificant effect b 1000 m
Ps. fluorescens Total inhibition by 2000 ppm for > 42 days
3756 (counts
undetectable).
No si ificant effect with 1000 m
Salmonella Typhimurium Reduction in final count by 2000 ppm (106
cf 109). After 8 d
S29 counts drop, undetectable byl8 d.
No effect with 1000 m
S. cerevisiae 9763 No effect with 2000 m
5
Table 9: Summarised results of cooked sausage meat slurry at 8 °C
[TSM010605]
Strain Result
B. cereus 204 No total inhibition by 1000 ppm, 106
reached at 7 d
Controls reached 106 b 5 d.
L. rnonocytogenes No total inhibition by 1000 ppm, 106
272 reached at 7 d
Controls reached 106 b 7 d
Br. thermosphacta No significant effect with 500 and 100
ppm
CRA78884
Lb. sake A10 No effect with 500 and 1000 m
Salm Typhimurium Total inhibition by 500 for 25 d
S29
Total inhibition by 1000 ppm for > 3
8 d
Slow owth in controls, reached 106 b
19 d
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31
Table 10: Summary of results of trial in minced beef slurry at 8 °C
[TCM010619]
Strain Result
B. cereus 204 2000 ppm delayed growth to 106 by 7
d.
1000 m extended la hase.
L. monocytogenes . No si ificant inhibition b 2000 m
272
Br. thermosphacta No effect with 2000 ppm
CRA78883
Lb. sake A10 No effect with 2000 m
Salm Typhimurium Total inhibition by 2000 ppm for >
S29 44 d
Slow owth in controls, reached 106
b 13 d
Ps. fluorescens . Total inhibition by 2000 ppm for 27
3756 d
1000 ppm delayed growth by 17 d
Controls reached 106 b 15 d
Table 11: Summary of results of trial in minced chicken slurry at 8 °C
[TCC010704]
Strain Results after 13 d incubation
B. cereus 204 Partial inhibition by 2000 ppm
Controls reached 106 b 5 d
L. monocytogenes. Sli ht inhibition b 2000 m onl
272
Salm TyphimuriumTotal inhibition by 1000 ppm for 25d
S29
Total inhibition by 2000 ppm for >40 d
Controls not reached 106 b 14 d
Ps. fluorescens No total inhibition by 2000 ppm, 106 reached
327 at 10 d
Controls reached 106 b 3 d
Table 12: Results of raw meat with added at 8°C. [TRM010521]
Date Day Viable counts
(cfu/ )
0 m APP 2000 m APP
21/5 0 5.6x10 9.3x10
22/5 1 2.0 x 10 2.2 x 10~
23/5 2 2.7 x 10a 1.5 x 10~
24/5 3 9.7 x 10a 5.0 x 10~
Table 13: Summary of test results for activity of 2000 ppm APP in food systems
Food/test systemsEffect on 00 m APP
rowth of
tar et or
anism b 20
Total inhibitionPartial inhibitionNo inhibition
Lab media at optimumLb. sake B. cereus Most yeasts
temperature Ps. fluorescensL. monocytogenes(enhanced growth)
Y. enterocoliticaBr. thermosphacta
Salmonella All Clostridium
s s
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32
Chicken soup at B. cereus S cerevisiae
8 C (1000
pH 5.8 L. monocytogerzes ppm)
Ps. fluorescens
Salrnonella
s
Chicken soup at B. cereus L. naonocytogenesS. cerevisiae
20 C
H 5.8 Ps. uorescensSalmonella
Apple juice at - Z. bailiff Candida
ambient
pH 3.4 S. cerevisiaeparapsilosis
H. uvarum
Rh. mucilaginosa
B ssochlam
s ulva
Milk at 20 C - Ps. fluorescensL. monocytogenes
pH 6.65 B. cereus Br. thermosphacta
Lb. sake
Milk at 8 C Ps. fluorescensBr. thermosphactaL. monocytogenes
pH 6.65 Lb. sake
B, cereus
Cooked sausage S. TyphimuriumB. cereus Br. thermosphacta
meat (1000)
slurry at 8 C (1000) L. monocytog.(1000)
H 6.2 (1000) Lb. sake (1000)
Cooked minced beefS. TyphimuriumB. cereus Br. thermosphacta
slurry at 8 C Ps, fluorescens Lb. sake
H 6.24 (L. monoc to
enes)
Cooked minced chickenS. TyphimuriumPs. fluorescens(L. monocytogenes)
slurry at 8 C B. cereus
H 6.42
Raw uninoculated - TAVC
meat
at8C. H5.3
Raw inoculated - TAVC
meat at
8 C. pH 5.3 L. monocytogenes
Salmonella
Typhimurium
Br. thermos
hacta
Various modifications and variations of the described methods and system of
the
invention will be apparent to those skilled in the art without departing from
the scope and
spirit of the invention. Modifications of the described modes for carrying out
the
invention which are obvious to those skilled in the relevant are, or related
fields, are thus
intended to fall within the scope of the following claims.
